Fluorescent substrates for Detecting organophosphatase enzyme activity

ABSTRACT

Disclosed are compounds of the formula (I): wherein R 3 , R 4 , R 5 , R 9 , and R 10  are selected from the group consisting of H and groups or atoms other than H, and R 6  and R 8  are halo or hydrogen; X 1 , X 2 , and X 3  are independently O or S; provided that R 9  and R 10  are not simultaneously H, when all of X 1 , X 2 , and X 3  are O; and of the formula (II) wherein R 11 -R 14  are selected from the group consisting of H and groups or atoms other than H; X 4 -X 9  are independently O or S; n and m are 0 or 1 but m and n cannot be 0 simultaneously; R 15 -R 24  can be H or any substituent so long as the compound of formula II upon hydrolysis provides a fluorescent compound. These compounds are useful as substrates with high specificity for organophosphatase particularly human paraoxonase and bacterial organophosphorus hydrolase. Also disclosed is a method for detecting and/or measuring the paraoxonase activity in a fluid comprising contacting the fluid with a fluorescent substrate and measuring the fluorescence of the fluorescent product formed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication Nos. 60/463,317, filed Apr. 17, 2003 and 60/487,935, filedJul. 18, 2003, the disclosures of which are incorporated by reference.

FIELD OF THE INVENTION

This invention relates to certain fluorescent substrates and a methodfor detecting organophosphatase activity in general, and paraoxonaseactivity specifically and in particular in biological fluids such asblood and serum, through the use of such fluorescent substrates.

BACKGROUND OF THE INVENTION

Enzymatic degradation of organophosphates (OPs) is performed byspecialized enzymes including bacterial organophosphorus hydrolase (OPH)and mammalian paraoxonase. Paraoxonase also referred to as, arylesterase(EC 3.1.1.2) is a 43 kDa molecular weight calcium dependent esterhydrolase that catalyses the hydrolysis of a broad range of esters suchas OPs, and unsaturated aliphatic and aromatic carboxylic esters. Itsname derives from the ability of this protein to hydrolyze paraoxon, thetoxic metabolite of the insecticide parathion. In addition to paraoxon,paraoxonase is able to detoxify a number of other insecticides, e.g.diazonin, as well as the potent nerve gases sarin and soman that targetacetylcholinesterase (AChE). The paraoxonase gene (PON) family consistsof at least three members: PON1, PON2 and PON3, which are located on thehuman 7q21.3-22.1 chromosome. No significant endogenous expression ofPON2 and PON3 genes has been detected. Most PON1 expression takes placein the human liver; from there the protein is secreted into blood whereit circulates associated with high density lipoprotein (HDL) particles.Paraoxonase has the unusual property that the mature protein retains itshydrophobic N-terminal signal peptide, which is used as an anchor forassociation with HDL. The enzyme has three potential N-linked sites andcarbohydrate accounts for approximately 16% of its molecular mass.

There is a significant variation in paraoxonase activity in the humanpopulation, which is a result of polymorphism in the PON1 promoter thatleads to different levels of expression, as well as polymorphism in genesequence that leads to allele forms of protein with different specificactivity. Both types of polymorphisms are quite common among the humanpopulation generating a range of paraoxonase serum activity in thepopulation. The apparent molecular mass of serum paraoxonase varies asthe result of heterogeneous glycosylation.

Neither the function nor natural substrate(s) for paraoxonase have yetbeen identified. One possible substrate is oxidized low densitylipoprotein (LDL) [1-3]. Paraoxonase has been shown both to preventformation of oxidized LDL and to hydrolyze LDL-derived oxidizedphospholipids. Since accumulation of oxidized LDL is one of the keyfactors in development of atherosclerosis, paraoxonase activity maycorrelate with development of this disease. For example, Shih et aldemonstrated that PON1−/− mice were extremely sensitive to diet-inducedatherosclerosis in comparison with wild type mice. Since there is asignificant variation in paraoxonase activity among the population,evaluation of paraoxonase levels of individuals may have a significantdiagnostic value, predicting the chances, development and prognosis ofatherosclerosis.

Another possible natural substrate is lipopolysaccharide (LPS) ormediators of septic shock. It has been shown that high densitylipoprotein (HDL) can inactivate LPS [4]. Moreover, intraperitonealinjection of mice before and up to 2 hours after LPS administrationafforded protection against septic shock [5]. In addition, PON-1knockout mice are extremely sensitive to LPS [6].

Paraoxonase is able to hydrolyze a number of OP toxins in vitro, and theability of paraoxonase to protect animals in acute OP poisoning has beenextensively studied. Injection of purified paraoxonase protected animalsagainst OP toxicity [7, 8]. Further proof of the ability of paraoxonaseto protect animals has been obtained from studies on PON1 “knock-out”mice. Destruction of the PON1 gene by knock-out technology creates micethat lack paraoxonase. Compared to wild type littermates, PON1 deficientmice were extremely sensitive to the toxic effects of chlorpyrifos, anOP. Thus, monitoring of paraoxonase activity may help to evaluate aperson's ability to withstand OP poisoning associated with deployment ofchemical weapons.

Consequently, monitoring blood levels of paraoxonase may be used toidentify, a predisposition to atherosclerosis, sepsis and OP poisoning.However, the absence of a robust test for detection of paraoxonaselevels in blood has significantly delayed progress in studying thediagnostic value of paraoxonase. There are two major options fordetection of this enzyme activity. The first is a change in opticaldensity and the second the generation of a fluorescent product.

Currently, the most common substrates for paraoxonase used in researchare paraoxon and phenylacetate. Paraoxonase catalyzed hydrolyses ofparaoxon leads to release of nitrophenol, which can be detected bymonitoring adsorption at 405 nm. This reaction is used to measureparaoxonase activity in fundamental and clinical research. The maindisadvantages of this substrate are the low Vmax of hydrolysis, whichresults in relatively low sensitivity and, due to its toxicity, paraoxonrequires special handling conditions. The arylesterase activity ofparaoxonase is usually measured through hydrolysis of phenylacetate.This reaction has a much higher Vmax, than the Vmax of paraoxonhydrolysis; however, phenylacetate is also hydrolyzed by a number ofother esterases in cell extracts and serum samples, which significantlydecreases the specificity of detection. In addition, the detection ofphenylacetate hydrolysis is based on monitoring adsorption at 270 nmmaking paraoxonase detection difficult, or impossible, in protein richsolutions or in extracts containing detergents like Triton X-100.

The OPH gene was originally found in two soil microorganisms,Pseudomonas diminuta and Flavobacterium sp. It has been suggested thatthis enzyme evolved recently in these bacteria in response to industrialsoil contamination with organophosphate compounds. Like paraoxonase, OPHcatalyzes a broad range of organophosphate esters including sarin andVX. Due to this activity these organisms may have additional utility indecontamination of OPs in the environment. In this context a sensitiveand robust assay would be necessary to confirm expression of OPH in thepresence of a large excess of phosphatase activity. Thus, it isessential that the substrate has very little or no affinity forphosphatases.

The foregoing shows that there exists a need for detectingorganophosphatase activity including paraoxonase with high specificityand sensitivity. There exists a need for substrates with highspecificity for OPH and paraoxonase. The advantages of the presentinvention as well as inventive features will be apparent from thedetailed description of the embodiments of the invention providedherein.

BRIEF SUMMARY OF THE INVENTION

The foregoing needs have been fulfilled to a great extent. The presentinvention provides highly sensitive and specific fluorescent substrates.In accordance with an embodiment, the present invention providescompounds of the formula (I):

wherein R³, R⁴, R⁵, R⁹, and R¹⁰ are selected from the group consistingof H and groups or atoms other than H, and R⁶ and R⁸ are halo orhydrogen; X¹, X², and X³ are independently O or S; provided that R⁹ andR¹⁰ are not simultaneously H, when all of X¹, X², and X³ are O.

In accordance with another embodiment, the present invention providecompound of the formula II:

wherein R¹¹-R¹⁴ are selected from the group consisting of H and groupsor atoms other than H; X⁴-X⁹ are independently O or S. n and m are 0 or1 but m and n cannot be 0 simultaneously. R¹⁵-R²⁴ can be H or anysubstituent so long as the compound of formula II upon hydrolysisprovides a fluorescent compound.

The present invention also provides a method for detecting and/ormeasuring the organophosphatases and particularly paraoxonase activityin a fluid comprising contacting the fluid with a fluorescent substrateand measuring the fluorescence of the fluorescent product formed.

The fluorescent substrates of this invention are specific fororganophosphatases including paraoxonase and, when hydrolyzed, releasehighly fluorescent products which can be measured at, for example, anemission wavelength of 460 nm following excitation at a wavelength of355 nm for structures based on the coumarin structure and emission of520 nm following excitation at 488 nm for fluorescein-based structures.In comparison with the other substrates used for the detection ofparaoxonase, these have significantly higher sensitivity andspecificity. The substrates of the present invention facilitate largethroughput methods for the detection and quantitation of this enzyme'sactivity. Such methods may be used for detection of paraoxonase as adiagnostic marker for prediction of atherosclerosis development, sepsisand sensitivity to OPs.

The substrates are useful for detecting and quantifying paraoxonaseactivity in samples of biological fluids such as blood. Measurement ofblood paraoxonase activity may be useful as an indicator ofcardiovascular disease and sensitivity to OP poisoning. Also provided isa method for detecting the activity of paraoxonase in an environmentalsample. Such samples may include those which have been treated withparaoxonase to decontaminate OPs. Also provided is a method for studyingthe basic properties of paraoxonase by using these substrates asresearch reagents. Also provided is a method of assaying for thepresence of OPs through the specific inhibition of substrate inducedfluorescence. The substrates of the present invention have one or morethan one advantage; e.g., high specificity for paraoxonase; highsensitivity fluorescent detection and a significant Vmax of reactionmakes it at least 10-20 times more sensitive than any other knownsubstrate for paraoxonase detection. Consequently, the substrates mayhave a significant practical use in different areas of medicine anddetection of nerve gas poisons.

The substrates are useful for detecting and quantifying OPH activity inenvironmental samples such as soil extracts or swabs. Such samples mayinclude those which have been treated with OPH to decontaminate OPs.Also provided is a method for studying the basic properties of OPH byusing these substrates as research reagents. Also provided is a methodof assaying for the presence of OPs through the specific inhibition ofsubstrate induced fluorescence. The substrates of the present inventionhave one or more than one advantage; e.g., high specificity for OPH;high sensitivity fluorescent detection and a significant Vmax ofreaction makes it at least 10-20 times more sensitive than any otherknown substrate for OPH detection. Consequently, the substrates may havea significant practical use in different areas of medicine and detectionof nerve gas poisons.

The proposed substrates can be used for broad screening of paraoxonaseactivity in human blood. Paraoxonase levels in the blood correlates withresistance to organophosphate poisoning, development of atherosclerosis,ability to detoxify LPS and general liver malfunctions. The presentinvention provides an assay kit for paraoxonase detection andquantitation. Such a kit may be used for detection of paraoxonase as adiagnostic marker for prediction of atherosclerosis development. Thediagnostic prognosis of paraoxonase detection is comparable to, orbetter than, such blood markers as blood cholesterol level. Such a kitmay also be used for detection of paraoxonase as a diagnostic marker forprediction of sepsis development. Another potential use of a kit fordetection of paraoxonase is predicting the resistance to OP challengeswhich can have a significant value in a war against chemical terrorismor during combat where chemical weapons are utilized. It is alsoenvisaged that the kit may be used to confirm that protective levels ofparaoxonase have been achieved in war fighters following administrationof prophylactic levels of recombinant paraoxonase. Moreover, a rapid andsensitive method for detection of organophosphatase activity may beuseful for the detection of alternative substrates, e.g., nerve poisons,OP toxins and insecticides, present in environment samples. Suchalternative substrates for organophosphatase may be identified by theirability to compete for binding and hydrolysis of the substrates of thepresent invention.

The present invention further provides a method for selectivelydetecting organophosphatase in a sample suspected to containorganophosphatase and a phosphatase comprising contacting the samplewith a substrate of the invention, measuring the fluorescence of afluorescent product formed during the contacting; and correlating themeasured fluorescence with the activity of the organophosphatase enzyme.The spectrum of structures provides a method to discover differentorganophosphatases with different spectra of substrate specificities.

The present invention further provides a method for detecting and/ormeasuring the activity of organophosphatase enzyme immobilized on asupport comprising contacting the support with a substrate of theinvention, measuring the fluorescence of a fluorescent product formedduring the contacting; and correlating the measured fluorescence withthe activity of the organophosphatase enzyme.

While the invention has been described and disclosed below in connectionwith certain embodiments and procedures, it is not intended to limit theinvention to those specific embodiments. Rather it is intended to coverall such alternative embodiments and modifications as fall within thespirit and scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the detection of paraoxonase activity in serum samplesusing 7[diethyl-phosphor]-6,8-difluoro-4-methylumbelliferyl (DEPFMU) asa substrate. In this assay 10 μl of rabbit and, separately 10 μl ofmouse serum both previously diluted 10 fold with assay buffer, 20 mMTris.HCl, pH 8.0, 150 mM NaCl and 2 mM CaCl₂, were incubated with 100 μlof assay buffer containing 100 μM of DEPFMU. The rates of change offluorescence at 37° C. were monitored at 355/460.

FIG. 2 depicts a cell membrane associated production of fluorescence inPON1 transfected and non-transfected CHO cells using DEPFMU as thesubstrate. In this experiment Chinese hamster ovary (CHO) cells weretransfected using Fugene 6 reagent (Roche) with the expression vectorpHLSS122 containing human PON1 cDNA. Transfection was performed in 96well microliter plates. 48 hours after transfection, the cells werewashed twice with PBS and 100 μl of assay buffer containing 100 μm ofDEPFMU were added. Immediately after addition, the rates of change offluorescence at 37° C. were monitored at 355/460.

FIG. 3 depicts the measurement of the Km of DEPFMU for rabbitrecombinant paraoxonase. The rabbit recombinant paraoxonase wasexpressed in CHO cells and partially purified. v-A, v-B and v-C are thevelocities at 1/5, 1/10 and 1/20 dilutions of recombinant paraoxonase,respectively. Partially purified recombinant paraoxonase was mixed withsolutions containing different concentrations of DEPFMU as substrate.The time-course of DEPFMU hydrolysis 37° C. was monitored usingfluorescence reading at 355/460. The reciprocal of the Km of DEPFMU wasobtained from the intercept on the abscissa using a Lineweaver-Burk Plot(1/v) where v is the velocity of the reaction against 1/[S] where [S] isthe substrate concentration.

FIG. 4 depicts a comparison of the sensitivity of paraoxonase detectionusing paraoxon and DEPFMU based assays. An evaluation of the relativesensitivity of DEPFMU (A) and paraoxon (B) based assays for paraoxonaseassay was performed using 10 μl serial dilution of rabbit serum samplesincubated with 100 μl of assay buffer comprising 4 mM paraoxon or 100 μMDEPFMU in the assay buffer. After incubation, the optical density at 405was measured to detect paraoxon and fluorescence (355/460) was read forDEPFMU. The two standard deviations above background readings isassigned as the limits of reliable detection. As can be seen from thefigure, paraoxonase activity can be reliably detected with more that1:10,000 dilution in the DEPFMU based assay, while less than 1:1,000 isrequired for detection with paraoxon based assay.

FIG. 5 depicts the hydrolysis of the DEPFMU by PON1 mutants.

FIG. 6A depicts the hydrolysis data for a substrate of the presentinvention, DEPFMU. FIG. 6B depicts the hydrolysis data for6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). See Example 8.

FIG. 7 depicts the hydrolysis of the fluorescein diphosphate tetra ethylester (FDPTEE) by bacterial OPH.

FIG. 8 depicts the hydrolysis of FDPTEE by normal mice contrasted withPON1 knock out mice.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention provides a compound of the formula I:

wherein R³, R⁴, R⁵, R⁹, and R¹⁰ are selected from the group consistingof H and groups or atoms other than H, and R⁶ and R⁸ are halo orhydrogen; X¹, X², and X³ are independently O or S; provided that R⁹ andR¹⁰ are not simultaneously H, when all of X¹, X², and X³ are O.

In accordance with an embodiment of the invention in formula I, R³, R⁴,and R⁵ are selected from the group consisting of H, hydroxyl, cyano,nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl,sulfinyl, sulfomethyl, a salt of sulfomethyl, thiocyanato, aldehydro,keto, carbamoyl, urethane, ureido, guanidino, C₁-C₆ alkylamino, C₁-C₆acylamino, C₁-C₆ alkylamido, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylthio,C₅-C₈ cycloalkyl, C₁-C₆ haloalkyl, C₁-C₆ perfluoroalkyl, formyl,carboxamide of the formula —(C═O)NR¹R² where R¹ and R² are independentlyH, alkyl having 1-6 carbon atoms, an aryl, or R¹ and R² taken togetherform a saturated 5- or 6-membered ring having the formula—(CH₂)₂-M-(CH₂)₂— where the ring moiety M is a single bond, an oxygenatom, a methylene group, or the secondary amine —NR⁷— where R⁷ is H oralkyl having 1-6 carbon atoms, C₅-C₈ halocycloalkyl, C₁-C₆ hydroxyalkyl,C₅-C₈ hydroxycycloalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, C₂-C₆ alkoxycarbonyl,C₂-C₆ alkoxycarbonyl C₁-C₆ alkyl, carboxy C₁-C₆ alkyl, carboxy C₁-C₆alkoxy, dicarboxy C₁-C₆ alkyl, dicarboxy C₁-C₆ alkoxy, C₂-C₆ cyanoalkyl,phosphono C₁-C₆ alkyl, phosphoryl C₁-C₆ alkyl, mono-, di-, andtrisaccharides, nucleic acids, oligonucleotides, amino acids, peptides,and proteins, and C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, arylcarbonyl, andheteroaryl, which may be optionally substituted with a substituentselected from the group consisting of hydroxyl, cyano, nitro, halo,amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl,thiocyanato, aldehydro, keto, carbamoyl, urethane, ureido, andguanidino; R⁹ and R¹⁰ are selected from the group consisting of H, C₁-C₆alkyl, C₅-C₈ cycloalkyl, C₁-C₆ haloalkyl, C₁-C₆ perfluoroalkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, aryl, arylcarbonyl, heteroaryl, C₁-C₆aminoalkyl, C₅-C₈ cycloalkyl, C₁-C₆ haloalkyl, C₅-C₈ halocycloalkyl,C₁-C₆ hydroxyalkyl, C₅-C₈ hydroxycycloalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl,C₂-C₆ alkoxycarbonyl, C₂-C₆ alkoxycarbonyl C₁-C₆ alkyl, carboxy C₁-C₆alkyl, carboxy C₁-C₆ alkoxy, dicarboxy C₁-C₆ alkyl, dicarboxy C₁-C₆alkoxy, C₂-C₆ cyanoalkyl, phosphono C₁-C₆ alkyl, phosphoryl C₁-C₆ alkyl,mono-, di-, and trisaccharides, nucleic acids, oligonucleotides, aminoacids, peptides, and proteins, and C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl,and heteroaryl, which may be optionally substituted with a substituentselected from the group consisting of hydroxyl, cyano, nitro, halo,amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl,thiocyanato, aldehydro, keto, carbamoyl, urethane, ureido, andguanidino; and R⁶ and R⁸ are halo, particularly fluoro.

In accordance with an embodiment of the invention in formula I, R⁴ isselected from the group consisting of H, hydroxyl, cyano, nitro, halo,amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl,sulfomethyl, salt of sulfomethyl, thiocyanato, aldehydro, keto,carbamoyl, urethane, ureido, guanidino, C₁-C₆ alkylamino, C₁-C₆acylamino, C₁-C₆ alkylamido, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆perfluoroalkyl, halomethyl, C₁-C₆ alkylthio, C₅-C₈ cycloalkyl, C₁-C₆haloalkyl, C₅-C₈ halocycloalkyl, C₁-C₆ hydroxyalkyl, C₅-C₈hydroxycycloalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, C₂-C₆ alkoxycarbonyl, C₂-C₆alkoxycarbonyl C₁-C₆ alkyl, carboxy C₁-C₆ alkyl, carboxy C₁-C₆ alkoxy,dicarboxy C₁-C₆ alkyl, dicarboxy C₁-C₆ alkoxy, C₂-C₆ cyanoalkyl,phosphono C₁-C₆ alkyl, phosphoryl C₁-C₆ alkyl, mono-, di-, andtrisaccharides, nucleic acids, oligonucleotides, amino acids, peptides,and proteins, and C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, arylcarbonyl, andheteroaryl, which may be optionally substituted with a substituentselected from the group consisting of hydroxyl, cyano, nitro, halo,amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl,thiocyanato, aldehydro, keto, carbamoyl, urethane, ureido, andguanidino. In a preferred embodiment, R⁴ is selected from the groupconsisting of H, cyano, sulfomethyl, salt of sulfomethyl, aryl, C₁-C₆alkyl, C₁-C₆ alkoxy, and C₁-C₆ perfluoroalkyl, more preferably C₁-C₆alkyl, for example, methyl.

In accordance with an embodiment of the invention in formula I, R⁹ andR¹⁰ are selected from the group consisting of H, C₁-C₆ alkyl, C₅-C₈cycloalkyl, C₁-C₆ haloalkyl, C₁-C₆ perfluoroalkyl, C₂-C₆ alkenyl, andC₂-C₆ alkynyl, and aryl, arylcarbonyl, and heteroaryl, which may beoptionally substituted with a substituent selected from the groupconsisting of hydroxyl, cyano, nitro, halo, amino, amido, azido, acetal,ketal, imido, sulfo, sulfonyl, sulfinyl, thiocyanato, aldehydro, keto,carbamoyl, urethane, ureido, and guanidino; and X¹, X², and X³ are O orS, preferably O.

In a preferred embodiment of formula I, R⁹ and R¹⁰ are selected from thegroup consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl,and heteroaryl, more preferably from the group consisting of H, C₁-C₆alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl. In a further preferredembodiment, R⁹ and R¹⁰ are selected from the group consisting of C₁-C₆alkyl, for example, R⁹ and R¹⁰ are ethyl.

In accordance with another embodiment of formula I, R³ is selected fromthe group consisting of H, cyano, C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, and heteroaryl, formyl, carboxamideof the formula —(C═O)NR¹R² where R¹ and R² are independently H, alkylhaving 1-6 carbon atoms, an aryl, or R¹ and R² taken together form asaturated 5- or 6-membered ring having the formula —(CH₂)₂-M-(CH₂)₂—where the ring moiety M is a single bond, an oxygen atom, a methylenegroup, or the secondary amine —NR⁷— where R⁷ is H or alkyl having 1-6carbon atoms.

In accordance with an embodiment of formula I, R⁵ is H or C₁-C₆ alkoxy,preferably H. In accordance with a preferred embodiment of formula I, R⁶and R⁸ are fluoro. Examples of preferred compounds include those whereinX¹, X², and X³ are O or S, more preferably O, R⁹ and R¹⁰ are ethyl, R⁴is methyl, R⁶ and R⁸ are fluoro, and R³ and R⁵ are H. Specific examplesof the compound of formula I are those wherein R⁹ and R¹⁰ are ethyl, R⁴is methyl, R⁶ and R⁸ are fluoro, and X¹, X², and X³ are O; and thosewherein X¹ and X² are O, X³ is S, R⁶ and R⁸ are H; R⁹ and R¹⁰ are ethyl,and R⁴ is methyl.

The compounds of the present invention can be prepared by any suitablemethod, for example, by following methods generally known in the art;see, e.g., U.S. Pat. Nos. 4,659,657; 5,428,059; 5,830,912; and6,416,970; and U.S. patent application publication No. US 2003/0032080A1; the disclosures of which are incorporated by reference. For example,the appropriate 7-hydroxycoumarin can be reacted with a suitablephosphate (or thiophosphate) ester compound to obtain the desired esteror thioester. For the preparation of dye conjugates, see G. T.Hermanson, Bioconjugate Techniques (Academic Press 1996).

The present invention provides a compound of the formula II:

wherein R¹¹-R¹⁴ are selected from the group consisting of H and groupsor atoms other than H; X⁴-X⁹ are independently O or S. m and n are 0 or1 but m and n cannot be 0 simultaneously. R¹⁵-R²⁴ can be H or anysubstituent so long as the compound of formula II upon hydrolysis, e.g.,of the P—X⁶ and/or P—X⁹ bonds, provides a fluorescent compound. When mor n is 0, the substituent at that position is H.

In an embodiment of the formula II, R¹¹-R¹⁴ are independently selectedfrom the group consisting of H, C₁-C₆ alkyl, C₅-C₈ cycloalkyl, C₁-C₆haloalkyl, C₁-C₆ perfluoroalkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl, andaryl, arylcarbonyl, and heteroaryl, which may be optionally substitutedwith a substituent selected from the group consisting of hydroxyl,cyano, nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo,sulfonyl, sulfinyl, thiocyanato, aldehydro, keto, carbamoyl, urethane,ureido, and guanidino; and X⁴-X⁹ are independently O or S, preferably O.In a preferred embodiment, m and n are 1.

In accordance with an embodiment of the invention in formula II, R¹⁵-R²⁴are independently selected from the group consisting of H, hydroxyl,cyano, nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo,sulfonyl, sulfinyl, sulfomethyl, a salt of sulfomethyl, thiocyanato,aldehydro, keto, carbamoyl, urethane, ureido, guanidino,C₁-C₆alkylamino, C₁-C₆ acylamino, C₁-C₆ alkylamido, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ alkylthio, C₅-C₈ cycloalkyl, C₁-C₆ haloalkyl, C₁-C₆perfluoroalkyl, formyl, carboxamide of the formula —(C═O)NR¹R² where R¹and R² are independently H, alkyl having 1-6 carbon atoms, an aryl, orR¹ and R² taken together form a saturated 5- or 6-membered ring havingthe formula —(CH₂)₂-M-(CH₂)₂— where the ring moiety M is a single bond,an oxygen atom, a methylene group, or the secondary amine —NR⁷— where R⁷is H or alkyl having 1-6 carbon atoms, C₅-C₈ halocycloalkyl, C₁-C₆hydroxyalkyl, C₅-C₈ hydroxycycloalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl, C₂-C₆alkoxycarbonyl, C₂-C₆ alkoxycarbonyl C₁-C₆ alkyl, carboxy C₁-C₆ alkyl,carboxy C₁-C₆ alkoxy, dicarboxy C₁-C₆ alkyl, dicarboxy C₁-C₆ alkoxy,C₂-C₆ cyanoalkyl, phosphono C₁-C₆ alkyl, phosphoryl C₁-C₆ alkyl, mono-,di-, and trisaccharides, nucleic acids, oligonucleotides, amino acids,peptides, and proteins, and C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl,arylcarbonyl, and heteroaryl, which may be optionally substituted with asubstituent selected from the group consisting of hydroxyl, cyano,nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl,sulfinyl, thiocyanato, aldehydro, keto, carbamoyl, urethane, ureido, andguanidino.

In a preferred embodiment of the compound of formula II, R¹¹-R¹⁴ areindependently selected from the group consisting of H, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, and heteroaryl, more preferably fromthe group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆alkynyl. In a further preferred embodiment, R¹¹-R¹⁴ are independentlyselected from the group consisting of C₁-C₆ alkyl, for example, R¹¹-R¹⁴are ethyl, and m and n are 1. Specific examples are a compound whereinX⁴-X⁹ are O, R¹⁵-R²⁴ are H, R¹¹-R¹⁴ are ethyl; and m and n are 1 and acompound wherein X⁴, X⁵, X⁷, and X⁸ are O; X⁶ and X⁹ are S; R¹⁵-R²⁴ areH; R¹¹-R¹⁴ are ethyl; and m and n are 1.

In the compounds of formula I and II, aryl is a 1-3 aromatic ringcontaining group, preferably phenyl; heteroaryl is a 5- or 6-memberedaromatic heterocycle that is optionally fused to an additional6-membered aromatic ring or to one or more heteroaromatic ringcontaining 1-3 heteroatoms selected from the group consisting of O, N,and S. Examples of heteroaryl include pyrrole, thiophene, furan,oxazole, isooxazole, or imidazole, benzoxazole, benzothiazole,benzimidazole, benzofuran, and indole.

The compounds of formula II can be prepared by any suitable method. Forexample, fluorescein diphosphate tetraethyl ester can be prepared asshown in Example 10.

In an embodiment, the compounds of the present invention can be linkedor conjugated to other molecules, groups, or substance, e.g., a dye, areactive group, an antibody, or a solid support.

“Assay Buffer” is the buffer used in the detection of paraoxonaseactivity and is composed of 20 mM Tris.HCl, 150 mM NaCl and 2 mM CaCl₂at pH 8.0 at 25° C.

“Biological fluid” is a sample of a fluid originating from a biologicalsource. Examples of biological fluids include, but are not limited toblood, blood-derived compositions, serum, cerebrospinal fluid, urine,saliva, milk, ductal fluid, tears, semen, cell or tissue extracts,culture medium from the expression of paraoxonase or mutations ofparaoxonase, samples arising from the fractionation of paraoxonase orHDL from biological samples.

“DEPFMU” is the abbreviation for7-diethyl-phospho-6,8-difluoro-4-methylumbelliferyl, a chemical compoundthat is one of the newly invented fluorogenic substrates for detectionof paraoxonase activity

“Environmental sample” is a sample obtained from the environment forpurposes of detection of paraoxonase or OPs. It may be soil, water, airor any other material obtained natural environment.

“FDPTEE” is the abbreviation for fluorescein diphosphate tetraethylester.

“OP” is the abbreviation for organophosphate, which includes a varietyof organic compounds that contain phosphorus and often have intenseneurotoxic activities. This includes such compounds as sarin and soman,which were originally developed as nerve gases, as well as others widelyused as insecticides and fire retardants.

OPH refers to the protein encoded by organophosphorus hydrolase which isexpressed by two soil dwelling bacteria, Pseudomonas diminuta andFlavobacterium sp. It hydrolyses a number of organic esters includingparaoxon.

“Paraoxonase” refers to the protein encoded by PON1 gene. Paraoxonase isa serum protein that possesses enzymatic activity. It hydrolyzes anumber of organic and phospho-organic esters including paraoxon. Thephysiological function of paraoxonase is not known with certainty.

“PON1” refers to the gene encoding the protein known as paraoxonase, orarylesterase (EC 3.1.1.2). Paraoxonase is present in normal human plasmaand the cDNA, and genes encoding the human protein have been sequencedand characterized.

“Substrate for organophosphatase” refers to one of a number of chemicalcompounds that are hydrolyzed by OPH and/or paraoxonase. These includeDEPFMU, phenyl acetate, oxidized lipids and paraoxon.

“355/460” refers to an excitation wavelength of 355 nm and an emissionwavelength of 460 nm.

The present invention further provides a method for detecting and/ormeasuring the activity of organosphosphatase in a fluid comprisingcontacting the fluid with a compound of formula I, wherein R³-R⁶ andR⁸-R¹⁰ can be any atom or group and X¹, X², and X³ are independently Oor S; or of the formula II, wherein R¹¹-R¹⁴ are selected from the groupconsisting of H and groups or atoms other than H; X⁴-X⁹ areindependently O or S. n and m are 0 or 1 but m and n cannot be 0simultaneously. R¹⁵-R²⁴ can be H or any substituent so long as thecompound of formula II upon hydrolysis provides a fluorescent compound;measuring the fluorescence of a fluorescent product formed during thecontacting; and correlating the measured fluorescence with the activityof the paraoxonase enzyme. Any of the compounds described above inparagraphs [0029]-[0036] and [0038]-[0042] may be employed in the methodof the present invention.

In an embodiment of the method of detecting and/or measuring theactivity of paraoxonase, the compound of formula I can be one whereinR³, R⁴, R⁵, R⁹, and R¹⁰ are selected from the group consisting of H andgroups or atoms other than H, and R⁶ and R⁸ are halo. In an embodimentof the method of detecting and/or measuring the activity of paraoxonase,the compound of formula II can be one wherein X⁴-X⁹ are O, m and n are1, R¹¹-R²⁴ are H.

In accordance with an embodiment, the compounds described above can beused for detection of organophosphatase activity and specificallyparaoxonase activity in a biological fluid. Examples of biologicalfluids include, but are not limited to, blood, blood-derivedcompositions or serum, cerebrospinal fluid, urine, saliva, milk, ductalfluid, tears, semen, brain, artery, vein and gland extracts. Otherfluids may contain culture medium from the expression of paraoxonase ormutations of paraoxonase. Still further fluids may be taken from methodsand processes resulting in the fractionation of paraoxonase or HDL frombiological samples. In an embodiment, the fluid is an environmentalfluid, for example, an extract of soil, water, or swab.

In accordance with an embodiment, the compounds described above can beused for detection of organophosphatase activity and specifically OPHactivity in a biological fluid or environmental extract. Examples ofbiological fluids include, but are not limited to, blood, blood-derivedcompositions or serum, cerebrospinal fluid, urine, saliva, milk, ductalfluid, tears, semen, brain, artery, vein and gland extracts. Otherfluids may contain culture medium from the expression of OPH ormutations of OPH. Still further fluids may be taken from methods andprocesses resulting in the fractionation of OPH from biological samples.In an embodiment, the fluid is an environmental fluid, for example, anextract of soil, water, or swab.

In a further embodiment, the present invention provides a method forpredicting the existence of cardiovascular diseases. The presentinvention further provides a method for predicting a person'ssensitivity to OPs. The activity of paraoxonase as measured using thecompounds of the present invention can be used as a predictor ofcardiovascular disease and sensitivity to OPs.

In a further embodiment, the present invention provides a method forpredicting the potential for septic shock. The present invention furtherprovides a method for predicting a person's sensitivity to LPS. Theactivity of paraoxonase as measured using the compounds of the presentinvention can be used as a predictor of sensitivity to LPS.

In another embodiment, the present invention provides a method forevaluating and/or predicting the functional activity of preparations ofHDL. The paraoxonase activity measured by the fluorescence in accordancewith the present invention may be used for evaluation/prediction offunctional activity of preparations of high density lipoproteins.

In a further embodiment, detection of fluorescence is achieved using anexcitation wavelength and an emission wavelength. In a furtherembodiment, OPH and paraoxonase activity can be monitored using afluorimeter with an excitation wavelength at 355 nm and an emissionwavelength at 460 nm. Other assay formats include analysis of OPHactivity in gels after protein separation by electrophoresis or analysisof paraoxonase expression/secretion in live or dead cells embedded inlow melting point agarose, immunoblotting, western blot analysis, andfluorescent detection in situ with detection by microscopy, visualinspection, via film. Alternatively OPH or paraoxonase may beimmobilized on supports such as membranes, resins or dipsticks.

In a further embodiment, organophosphatase activity may be detected onthe surface of cells expressing paraoxonase activity using a cell sorter(e.g., fluorescence assisted cell sorter or FACS).

In a further embodiment the compounds of the present invention can beused to quantify the activity of OPases such as those associated withparaoxonase or variants of paraoxonase including natural variants orartificially created mutant forms of paraoxonase.

In a further embodiment the compounds of the present invention can beused to quantify the activity of organophosphatases such as thoseassociated with OPH or variants of OPH including natural variants orartificially created mutant forms of OPH.

In a further embodiment the presence of competing substrates (orcompounds) for organophosphatase can be identified by inhibition offluorescence in the presence of a substrate for OPH or paraoxonase. In apreferred embodiment, the substrate is DEPFMU and the competingsubstrate is an OP such as sarin or soman. In a further embodiment,paraoxonase is immobilized on a support and its activity is measured bythe production of a fluorescent signal. Environmental extracts includingwater and air, extracts of swabs are added to paraoxonase andalternative substrates identified by a decrease in fluorescence.Environmental extracts may be extracted with aqueous or organicsolvents, supercritical fluids, subcritical fluids, and the like.

In a further embodiment, OPH is immobilized on a support and itsactivity is measured by the production of a fluorescent signal.Environmental extracts including water and air, extracts of swabs areadded to OPH and alternative substrates identified by a decrease influorescence. Environmental extracts may be extracted with aqueous ororganic solvents, supercritical fluids, subcritical fluids, and thelike.

A wide variety of organic and inorganic polymers, both natural andsynthetic, may be employed as the material for immobilizingorganophosphatases such as OPH or paraoxonase. Illustrative polymersinclude polyethylene, polypropylene, polymethacrylate, polyacrylate,rayon, nylon, cellulose, nitrocellulose, and polyvinylidene fluoride.The immobilized organophosphatase can be quantified by contacting with acompound of the present invention.

In a further embodiment, the present invention provides a method formonitoring decontamination of the environment of OPs. The extract of thesoil treated with paraoxonase (for decontamination) can be contactedwith a compound of the present invention, and the fluorescence producedcan be an indication of the completeness of decontamination. In a stillfurther embodiment, the compound of the present invention can beemployed to identify soil-dwelling micro-organisms or plants whichexpress either OPH and/or paraoxonase.

In a further embodiment the organophosphatase is coupled to a secondarystructure such as an antibody with specificity for an alternative targetsuch that the secondary structure binds to its target to form aorganophosphatase-secondary structure: target complex. Theorganophosphatase may then be used as a reporter protein and thepresence of the organophosphatase-secondary structure: target complexidentified using the substrates or compounds of the present invention.

In a further embodiment, the PON1 gene is co-transfected with anotherprotein of interest and used as a reporter gene for expression of thetarget protein. In a still further embodiment, the PON1 gene is underthe control of different promoters and the fluorescent substrate used todetermine the activity of different promoters.

In a further embodiment, the OPH gene is co-transfected with anotherprotein of interest and used as a reporter gene for expression of thetarget protein. In a still further embodiment, the OPH gene is under thecontrol of different promoters and the fluorescent substrate used todetermine the activity of different promoters.

In a further embodiment the substrate is added to cells incubated withdifferent molecules that may up-regulate the PON1 promoter and hence theexpression of paraoxonase. Up-regulators of paraoxonase expression areidentified by the increase in fluorescent signal in the presence ofparaoxonase substrate. These regulators may affect signal transductionpathways that ultimately result in up-regulation of the gene promoter.

Unexpectedly, the replacement of two protons on the phosphate group of6,8-difluoro-4-methylumbelliferyl phosphate by ethyl groups (and otherlow molecular weight groups, e.g., hydrocarbon groups) generates a poorsubstrate for serum or cell-derived phosphatases but a good substratethat is selectively hydrolyzed by serum and recombinant paraoxonases andOPH. This is in marked contrast to phosphatase specific substrates, suchas 6,8-difluoro-4-methylumbelliferyl phosphate which is readilyhydrolyzed by phosphatases, but not by paraoxonase. Thus, DEPFMU may beused as a paraoxonase specific substrate which can accurately detectserum paraoxonase activity even in the presence endogenous phosphatase.Consequently, a major advantage of DEPFMU over prior art, is theunexpectedly low specificity of this substrate to phosphatase and itshigh specificity for organophosphatases such as OPH and paraoxonase.

The present invention further provides a method for detecting and/ormeasuring the activity of organophosphatase including paraoxonaseimmobilized on a support comprising contacting the support with any ofthe compounds of formula I or II; measuring the fluorescence of afluorescent product formed during the contacting; and correlating themeasured fluorescence with the activity of the paraoxonase enzyme. In anembodiment, the support is a membrane, resin, biosensor, microtiterplate, nanotube or dipstick, fiber, silicon chip, magnetic beads, anddifferent gels.

The present invention further provides a method for selectivelydetecting organophosphatase in a sample suspected to containorganophosphatase and a phosphatase comprising contacting the samplewith any of the compounds of formula I or II, e.g., the compound offormula I, wherein R³, R⁴, R⁵, R⁹, and R¹⁰ are selected from the groupconsisting of H and groups or atoms other than H, and R⁶ and R⁸ are haloor H; and the compound of formula II, wherein X⁴-X⁹ are O, m and n are1, R¹¹-R²⁴ are H; measuring the fluorescence of a fluorescent productformed during the contacting; and correlating the measured fluorescencewith the activity of the organophosphatase enzyme.

The present invention further provides a method for detecting and/ormeasuring the activity of organophosphatase enzyme immobilized on asupport comprising contacting the support with any of the compounds offormula I or II; e.g., the compound of formula I, wherein R³, R⁴, R⁵,R⁹, and R¹⁰ are selected from the group consisting of H and groups oratoms other than H, and R⁶ and R⁸ are halo or H; and the compound offormula wherein X⁴-X⁹ are O, m and n are 1, R¹¹-R²⁴ are H; measuring thefluorescence of a fluorescent product formed during the contacting; andcorrelating the measured fluorescence with the activity of theorganophosphatase enzyme.

The substrates of the present invention provide unexpected specificityand sensitivity for detection of OPases even in the presence of the highacid and alkaline phosphatase activities found in cellular extracts,plasma and sera. The fact that the substrates of the present inventionare even recognized by organophosphatases is surprising in view of thelarge size of the fluorogenic groups used relative to the knownsubstrates for these proteins. Thus, there is no data available a priorito suggest that such a bulky substrate would be accessible to the activesite of organophosphatases such as paraoxonase let alone selectivelyhydrolyzed by the protein.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the specificity of7-[diethyl-phospho]-,8-difluor-4-methylumbelliferyl towards paraoxonase.

The tested compounds included: 4-methylumbelliferyl acetate,4-methylumbelliferyl oleate, 4-methylumbelliferyl heptanoate,4-methylumbelliferyl palmitate,6,8-difluoro-4-methylumbelliferyl-octanoate,7-[diethyl-phospho]-6,8-difluoro-4-methylumbelliferyl [DEPFMU], andELF-97 palmitate (Molecular Probes, OR), fluorescein diphosphatetetraethyl ester, (3-carboxypropyl)-trimethylammonium chloride4-methylumbelliferyl ester, and7-benzyloxy-6,8-difluoro-4-methylumbelliferyl. All compounds wereevaluated for possible detection of paraoxonase activity usingrecombinant human paraoxonase, expressed in CHO cells. For expression ofrecombinant paraoxonase, human liver cDNA was obtained from Ambion Inc,(Austin, Tex.). Human PON1 cDNA was amplified by PCR with gene specificprimers, TRSSP 216 [AAGAATTCCACCATGGCGAAGCTGATTGCGCTC] [SEQ ID NO:1] andTRSSP 217 [AATCTAGATTAGAGCTCACAGTAAAGAGCTTTGTG] [SEQ ID NO:2] containingXba I and EcoRI restriction sites. Hassett C, Richter R J, Humbert R,Chapline C, Crabb J W, Omiecinski C J, Furlong C E. Characterization ofcDNA clones encoding rabbit and human serum paraoxonase: the matureprotein retains its signal sequence. Biochemistry, October 22;30(42):10141-9 (1991). Amplified PCR product containing PON1 cDNA wascloned into pBlueScript KS II vector (Stratagene, CA) as XbaI/EcoRIfragment, sequenced from T3 and T7 primers to confirm identity of DNAand then subcloned into an expression vector containing EF-1a promoterand GC-MSF poly (A) signal, forming expression vector pHLSS131. Theexpression vector DNA was propagated in E. coli, DH5a strain, andpurified using Quagen Maxiprep kit for plasmid purification (Quagen,CA). CHO cells were obtained from ATCC and cultivated according to therecommended conditions. CHO cells were transfected with the expressionvector using Fugene 6 reagent (Roche Diagnostic, Indianapolis, Ind.)according the manufacturer's manual. 48 hours after transfection, thelevel of PON1 expression was easily detectable by using standardsubstrates like paraoxon and phenylacetate. The test compounds werescreened for paraoxonase mediated hydrolysis by incubation withtransfected CHO cells. Fluorescent monitoring of the reaction wasperformed using SpectraMax GenimiXS fluorimeter, Molecular Devises Inc.(Sunnyvale, Calif.).

DEPFMU was specifically hydrolyzed by paraoxonase. After hydrolysis,highly fluorescent 6,8-difluoro-4-methylumbelliferyl is released. Sincethe DEPFMU itself does not possess any significant fluorescence,hydrolysis can be easily and safely monitored using any commercialfluorimeter with excitation at 355 nm and emission at 460 nm.

Example 2

This example illustrates an assay for detection of paraoxonase activity.DEPFMU may be used as a substrate for detection of serum derived as wellas recombinant paraoxonase expressed in cell cultures. The followingconditions have been used for the detection of paraoxonase in plasma.Samples of serum or plasma may be diluted 1 to 100 times in an assaybuffer. This assay was performed in a 96 well “Nunc-immuno” plate. Forexample, 10 μl of diluted rabbit serum was added to each well of a 96well plate containing 100 μl of assay buffer plus 100 μM of DEPFMU(stock DEPFMU was prepared as a 50 mM concentrate in dimethylformamideand stored at −20° C.). After thorough mixing the assay solutioncontaining plasma was incubated for 20 min. at 37° C. Hydrolysis ofDEPFMU was quantified by measuring the fluorescence level at 355/460using a commercial fluorimeter. The level of fluorescence correlateswith the level of 6,8-difluoro-4-methylumbelliferyl released from DEPFMUas result of paraoxonase mediated hydrolysis. The actual amount of6,8-difluoro-4-methylumbelliferyl released in the assay may becalibrated with a known amount of 6,8-difluoro-4-methylumbelliferyl. Anexample of paraoxonase detection in different serum samples is presentedin FIG. 1. Similar methods can be used for detection of recombinantparaoxonase expressed in cell culture.

Since a significant percentage of recombinant paraoxonase staysassociated with the cell membrane, methods were developed for evaluatingthe cell membrane associated paraoxonase. One such method harvests cellsexpressing paraoxonase, washes them twice with PBS (Dulbecco's PhosphateBuffer System) to remove endogenous paraoxonase which originates fromeither paraoxonase present in the growth medium or is synthesized andsecreted by the cells. Washed cells are pelleted by centrifugation andresuspended in PBS at a density of up to 10⁷ cells/ml. 10 μl of the cellsuspension is mixed with 100 μl of substrate solution per well of a 96well microtiter plate, incubated for 20 min. and the fluorescence levelmeasured as described above. As an option for this assay, adhesive cellsmay be plated into a microtiter plate a day or more before evaluation.The medium is then removed and the cells washed two times with PBS. 100μL of substrate solution is added and fluorescence monitored after 20 ormore minutes of incubation at 37° C. An example of the detection ofmembrane-associated paraoxonase is shown in FIG. 2. This shows thedetection of paraoxonase activity with DEPFMU for CHO cells transfectedwith PON1 versus background fluorescence using non-transfected cells.

Example 3

This example illustrates a method of detecting recombinant paraoxonaseactivity in CHO cells transfected with paraoxonase.

Human PON1 cDNA was recovered, cloned into pBlueScript KS II, andsequenced to confirm identity. For studies on the expression ofparaoxonase PON1, cDNA was subcloned in expression vector under thecontrol of the EF-1a promoter. CHO cells were transfected using Fugene 6reagent with expression vector pHLSS122, containing human PON1 cDNAunder EF-1a promoter in pBlueScript KS II cloning vector. Transfectionwith pBlueScript KS II vector DNA was performed for control CHO cells.Transfected cells were plated into 96 well tissue culture plates. 48hours after transfection, the wells were washed twice with PBS and 100μl of assay buffer containing 100 μM DEPFMU was added. Immediately afteraddition, the fluorescence of the wells was monitored using a 96 wellSpectraMax Gemini XS fluorescence reader at 355/460.

Data from this experiment is presented on FIG. 2. Mock-transfected CHOcells do not hydrolyze DEPFMU, whereas cells transfected with the vectorexpressing paraoxonase catalyses significant hydrolysis of thesubstrate. Since the only difference between the two cell populations isthat transfected cells express human paraoxonase activity whereas thecontrol cells do not. This further demonstrates that the substrates ofthe present invention are highly specific for paraoxonase and there areno significant levels of enzyme in normal cells which are able tohydrolyze DEPFMU.

Example 4

This example illustrates a method for measuring the Km of recombinantrabbit paraoxonase for DEPFMU.

CHO cells were transfected with the plasmid pHLSS131 expressing rabbitPON1 cDNA. See example 1 and 2 for additional details of transfectionand expression. After transfection the cells were propagated in growthmedium, harvested and paraoxonase was partially purified from the cellmembrane by extraction with 0.03% Tergitol (Sigma) in PBS. Extractedparaoxonase was separated from cells by centrifugation at 10000 g for 10min. The supernatant was considered as partially purified paraoxonase.10 μl of three different dilutions 1/5, 1/10 and 1/20 of recombinantparaoxonase (1/v-A, 1/v-B and 1/v-C) were incubated with 100 μl ofserially diluted DEPFMU as the substrate at 37° C. in a 96 wellmicro-titer plate. The time-course of DEFPMU hydrolysis was measured bymonitoring fluorescence every 5 min at 355/460 as described above. Dataon this experiment is presented in FIG. 3. Velocity of reaction wascalculated as the change in the level of fluorescence over time. Stablevelocity of reaction was observed up to 30 min of reaction. Actual Km ofparaoxonase for DEPFMU was calculated as 666 μM. For comparison the Kmof human paraoxonase for paraoxon is around 500 μM [9].

Example 5

This example provides a comparison of the sensitivity of paraoxon and acompound of the present invention as a substrate for paraoxonasedetection.

This experiment demonstrates the relative sensitivity of DEPFMU-basedassay compared with paraoxon based assays. 10 μl of serially dilutedsamples of rabbit serum were incubated for 30 min at 37° C. in thepresence of 100 μl of 4 mM paraoxon or 100 μl of 100 μM DEPFMU in theassay buffer described above. After incubation, the optical densitychange of paraoxon was measured at a wavelength of 405 nm and thefluorescence of DEPFMU hydrolysis was measured at 355/460. The data ispresented in FIG. 4. Reliable detection of paraoxonase activity wasconsidered to be greater than two standard deviation units abovebackground. This experiment demonstrates that the limit of detection ofenzyme activity for paraoxon-based assay was less than 1/1,000 dilutionof serum whereas that for DEPFMU was greater than a 1/10,000 folddilution. Thus, DEPFMU-based assays a significantly higher sensitivity.The sensitivity and velocity of fluorescent detection of DEPFMU wassub-optimal due to using a substrate concentration (100 μM), well belowthe Km of [˜666 μM], whereas the assay using paraoxon was well above theKm.

Example 6

This example illustrates the hydrolysis of DEPFMU and detection of PON1activity in CHO cells transfected with different PON1 mutants.

2×10⁵ CHO cells in a 96 well plate were transfected by 1 μg of DNA withFugene 6 reagent. DNA vector contained PON1 cDNA driven by EF-1promoter. Since different natural variants of PON1 have differentsubstrate specificity, four different variant/mutants of PON were used(145-6, 142-2, 131-10, and 122-7). 48 hours after transfection, cellswere washed with PBS and 100 μL of the DEPFMU substrate were added.Final concentration of the substrate was 40 μg/mL in 50 mM Tris bufferpH 8.0, 100 mM NaCl, and 1 mM CaCl₂. After 10 min. of incubation at 37C, fluorescence was measured at 355 nm excitation and 460 nm emission.The results are shown in FIG. 5. All four mutants hydrolyzed the DEPFMUwith very high efficiency. Spontaneous hydrolysis of substrate by CHOnon-transfected cells was about 3%. The control CHO cell did nothydrolyze the substrate.

Example 7

This example illustrates the sensitivity of detection provided by anembodiment of the present invention. We compared the sensitivity ofdetection of a serial dilution of OPH from 10 μg/ml to 0.5 ng/ml usingdifferent substrates including paraoxon and DEPFMU. 10 μl of OPHsolution was mixed with 100 μl of buffer containing 100 μM of DEPFMU, or1.2 mM of paraoxon. Samples were incubated for 30 min at 37° C. withchanges in optical properties monitored every 5 min. Assuming amolecular weight of 39,000 kDa and 100% purity and activity of theprotein as low as 1 femtomole (fmol) of OPH per well was reliablydetected. For paraoxon the level of reliable detection was around 100fmole of OPH per well. The catalytic rate (k_(cat)) of OPH mediatedhydrolysis was 1.5×10³ min⁻¹ for DEPFMU and 4×10⁴ min⁻¹ for paraoxon.Though the k_(cat) for paraoxon was more than 10 times higher than thek_(cat) for DEPFMU, the superior signal to noise ratio for the coumarinfluorophore over the optical change in nitrophenol, which was releasedafter paraoxon hydrolysis, makes the DEPFMU based assay systemapproximately 100 times more sensitive for OPH detection than paraoxon.The Km of DEPFMU for OPH was analyzed. For this experiment 10 μl of OPHsolution containing 25 fmoles of enzyme were mixed with 100 μl of 100 μMDEPFMU. The velocity of hydrolysis was constant only during the first 10min of reaction and decreases after 10-15 min. A decline in hydrolysiswas detected only after 25-30 min (data not shown) following OPHmediated hydrolysis of paraoxon. The Km of DEPFMU for OPH, was evaluatedusing concentrations of DEPFMU ranging from 290 μM to 2 μM. 10 μl ofsolution containing 25 fmoles of OPH were mixed with 100 μl of substratesolution. The reciprocal of the Km of DEPFMU was obtained from theintercept on the abscissa using a Lineweaver-Burk Plot (1/v) where v isthe velocity of the reaction against 1/[S] where [S] is the substrateconcentration. The apparent Km was calculated as 29 μM.

Example 8

This example illustrates a superior property of a substrate of thepresent invention, DEPFMU, relative to that of6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as substrate fordetection of paraoxonase. CHO cells were transfected using Fugene 6reagent with the expression vector pHLSS122, containing human PON1 cDNAunder EF-1a promoter in pBlueScript KS II cloning vector. Transfectionwith pBlueScript KS II vector DNA was performed for control CHO cells.Different plasmids 182, 178, 190, 184 188 and 186 containing differentmutants and natural variants of PON1 were used for expression.Transfected cells were plated into 96 well tissue culture plates. 48hours after transfection, the wells were washed twice with PBS and 100μl of assay buffer containing 100 μM DEPFMU or 100 μM DiFMUP was added.Immediately after addition, the fluorescence of the wells was monitoredusing a 96 well SpectraMax Gemini XS fluorescence reader at anexcitation wavelength of 355 nm and an emission wavelength of 460 nm.The data from this experiment is presented on FIGS. 6 a and 6 b. On FIG.6 a, DEPFMU hydrolysis by control and transfected cells wasdemonstrated. As it is seen from the figure, mock-transfected CHO cellsdo not hydrolyze DEPFMU, whereas cells transfected with the vectorexpressing paraoxonase catalyses significant hydrolysis of thesubstrate. FIG. 6 b demonstrates data on DiFMUP hydrolysis by controland transfected CHO cells. The control cells as well as experimentallytransfected cells do hydrolyze significant amount of DiFMUP, indicatingthere is significant PON1 independent hydrolysis of DiFMUP. Mostprobably this high level of hydrolysis is mediated by cell phosphatases,which are abundant in the majority of cell lines. The significantparaoxonase independent hydrolysis of DiFMUP precludes this substratebeing useful for the detection of paraoxonase.

Example 9

This example illustrates a method for the detection oforganophosphatase. Fluorescein diphosphate tetraethyl ester (FDPTEE) wasused as a substrate for detection of organophosphatase activity. Theexperiment was performed as described above in example 7. Briefly,serial dilution of OPH from 10 μg/ml to 0.5 ng/ml was prepared. 10 μl ofthe various dilution of OPH were mixed with 100 μl of buffer containing100 μM of FDPTEE. Samples were incubated for 30 min at 37° C. andfluorescence (Ex 488 nm/Em 520 nm) was monitored every 5 min. Theresults obtained are shown in FIG. 7. Assuming that the enzyme has amolecular weight of 39,000 kDa and is 100% pure and fully active as lowas 100 femtomole (fmol) of OPH per well was reliably detected, showingit to be applicable as a substrate for OPase.

The specificity of FDPTEE for paraoxonase was demonstrated using serasamples obtained from mice than had their PON1 gene destroyed throughembryonic stem cell technology (PON1 KO) (10). In these experiments, 10μl of sera samples from normal C57B1/6 mice and PON1 KO C57 Black/6 micewere diluted 1/10 with 150 mM NaCl and 20 mM Tris pH 8.0. 10 μl ofdiluted sera were mixed with 100 μl of 100 μM FDPTEE and incubated for30 min at 37° C. After incubation the level of fluorescence was measured(Ex 488 nm/Em 520 nm). The results obtained are shown in FIG. 8. Nofluorescence was detected in samples of sera obtained from PON1 KO mice,whereas significant fluorescence was detected in sera samples obtainedfrom normal C57Black mice. These data confirm that serum paraoxonase isthe enzyme responsible for FDPTEE hydrolysis and generation offluorescence signal in normal samples.

Example 10

This example illustrates a method of preparing an embodiment of thecompound of formula II, namely, fluorescein diphosphate tetraethylester. 2.5 g of fluorescein (7.5 mmol) is suspended in THF (60 mL) andanhydrous CH₂Cl₂ (mL). 3.1 g of 1H-tetrazole 3.1 g. (44 mmol) is addedand stirred at room temperature for ˜1.5 hours or until the reactionmixture becomes a transparent dark yellowish solution. The resultingsolution is cooled to 0° C. and 5.0 g of diethylN,N-diisopropylphosphoramidite (23 mmol) is added dropwise over a periodof 3-4 minutes to yield a very light yellow solution. The cooling bathis removed and the reaction stirred at room temperature until TLC showsno starting material remains (˜5 minutes). The reaction is cooled backto 0° C. R_(f) (fluorescein diphosphite, tetraethyl ester)=0.7-0.75, aquenching spot that becomes fluorescent yellow upon heating;hexanes/EtOAc (3:2) A solution of 3-chloroperoxybenzoic acid (MCPBA)(5.5 g, 32 mmol) in CH₂Cl₂/CHCl₃ (9:1, 50 mL) is prepared, and washedwith saturated NaCl (1×50 mL), followed by drying over Na₂SO₄. The driedsolution is added to the 0° C. reaction mixture to yield a cloudyyellowish solution. The cooling bath is removed and the reaction stirredat room temperature until TLC indicates completion (˜10 minutes). Thesolvent is removed from the reaction mixture in vacuo and the resultingyellow gum is dissolved in EtOAc (˜100 mL). The solution is washed with10-20% sodium thiosulfate/H₂O (1×100 mL), saturated NaHCO₃ (1×100 mL),and saturated NaCl (1×100 mL), and dried over Na₂SO₂. The solvent isremoved in vacuo. At this point, TLC in hexanes/EtOAc (3:2) shows twospots: R_(f)-0.65-0.7, a quenching spot that becomes fluorescent uponheating; and R_(f)-0.95-1.0, a quenching spot that does not fluoresceupon heating. 50 mL of methanol are added to the gum to precipitate thehigh-R_(f) material. The solid is removed by filtration, and methanol isremoved from the filtrate in vacuo. The resulting product is purified bycolumn chromatography under the following conditions:

Stationary Phase medium SiO₂ Mobile Phase CHC1₃ → 50% MeOH/CHC1₃About 50 mL of hexane is added to the resulting oily material to form asolid. The solid is collected with filtration and dried to constantweight in vacuo to yield FDP tetraethyl ester (fluorescein diphosphatetetraethyl ester) as a white sold. Actual yield 1.61 g (36%).R_(f)(VI)=0.25, a quenching spot that becomes fluorescent yellow uponheating; EtOAc/hexanes (2:1)

REFERENCES

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All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-56. (canceled)
 57. A compound of the formula II:

wherein R¹¹-R¹⁴ are selected from the group consisting of C₁-C₈ alkyl,C₅-C₈ cycloalkyl, C₁-C₆ haloalkyl, C₁-C₆ perfluoroalkyl, C₂-C₆ alkenyl,and C₂-C₆ alkynyl, and aryl, arylcarbonyl, and heteroaryl, which may beoptionally substituted with a substituent selected from the groupconsisting of hydroxyl, cyano, nitro, halo, amino, amido, azido, acetal,ketal, imido, sulfo, sulfonyl, sulfinyl, thiocyanato, aldehydro, keto,carbamoyl, urethane, ureido, and guanidino; X⁴-X⁹ are independently O orS; n and m are 0 or 1 but m and n cannot be 0 simultaneously; andR¹⁵-R²⁴ can be H or any substituent so long as the compound of formulaII upon hydrolysis provides a fluorescent compound.
 58. The compound ofclaim 57, wherein the hydrolysis takes place at the P—X⁶ and/or P—X⁹bonds.
 59. The compound of claim 57, wherein m and n are
 1. 60. Thecompound of claim 57, wherein R¹⁵-R²⁴ are independently selected fromthe group consisting of H, hydroxyl, cyano, nitro, halo, amino, amido,azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl, sulfomethyl, asalt of sulfomethyl, thiocyanato, aldehydro, keto, carbamoyl, urethane,ureido, guanidino, C₁-C₆ alkylamino, C₁-C₆ acylamino, C₁-C₆ alkylamido,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylthio, C₅-C₈ cycloalkyl, C₁-C₆haloalkyl, C₁-C₆ perfluoroalkyl, formyl, carboxamide of the formula—(C═O)NR¹R² where R¹ and R² are independently H, alkyl having 1-6 carbonatoms, an aryl, or R¹ and R² taken together form a saturated 5- or6-membered ring having the formula —(CH₂)₂-M-(CH₂)₂— where the ringmoiety M is a single bond, an oxygen atom, a methylene group, or thesecondary amine —NR⁷— where R⁷ is H or alkyl having 1-6 carbon atoms, anaryl, or R¹ and R² taken together form a saturated 5- or 6-membered ringhaving the formula —(CH₂)₂-M-(CH₂)₂— where the ring moiety M is a singlebond, an oxygen atom, a methylene group, or the secondary amine —NR⁷—where R⁷ is H or alkyl having 1-6 carbon atoms, C₅-C₈ halocycloalkyl,C₁-C₆ hydroxyalkyl, C₅-C₈ hydroxycycloalkyl, C₁-C₆ alkoxy C₁-C₆ alkyl,C₂-C₆ alkoxycarbonyl, C₂-C₆ alkoxycarbonyl C₁-C₆ alkyl, carboxy C₁-C₆alkyl, carboxy C₁-C₆ alkoxy, dicarboxy C₁-C₆ alkyl, dicarboxy C₁-C₆alkoxy, C₂-C₆ cyanoalkyl, phosphono C₁-C₆ alkyl, phosphoryl C₁-C₆ alkyl,mono-, di-, and trisaccharides, nucleic acids, oligonucleotides, aminoacids, peptides, and proteins, and C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl,arylcarbonyl, and heteroaryl, which may be optionally substituted with asubstituent selected from the group consisting of hydroxyl, cyano,nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl,sulfinyl, thiocyanato, aldehydro, keto, carbamoyl, urethane, ureido, andguanidino.
 61. The compound of claim 57, wherein R¹¹-R¹⁴ areindependently selected from the group consisting of C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, aryl, and heteroaryl.
 62. The compound of claim57, wherein R¹¹-R¹⁴ are independently selected from the group consistingof C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl.
 63. The compound ofclaim 57, wherein R¹¹-R¹⁴ groups are independently selected from thegroup consisting of C₁-C₆ alkyl.
 64. The compound of claim 57, whereinR¹¹-R¹⁴ is ethyl.
 65. A compound of formula II

wherein X⁴-X⁹ are O, R¹⁵-R²⁴ are H, R¹¹-R¹⁴ are ethyl; and m and nare
 1. 66. A compound of formula II:

wherein X⁴, X⁵, X⁷, and X⁸ are O; X⁶ and X⁹ are S; R¹⁵-R²⁴ are H;R¹¹-R¹⁴ are ethyl; and m and n are
 1. 67. A method for specifically andselectively detecting and/or measuring the activity of anorganophosphatase enzyme in a fluid, which contains at leastorganophosphatases and phosphatases, said method comprising:

(a) contacting the fluid with a compound of the formula II: whereinR¹¹-R¹⁴ are selected from the group consisting of H and groups or atomsother than H, X⁴-X⁹ are independently O or S, n and m are 0 or 1 but mand n cannot be 0 simultaneously, and R¹⁵-R²⁴ can be H or anysubstituent so long as the compound of formula II upon hydrolysisprovides a fluorescent product; (b) collecting the fluorescent product;(c) measuring the fluorescence of a fluorescent product formed duringthe contacting; and (d) correlating the measured fluorescence with theactivity of the organophosphatase enzyme.
 68. A method for selectivelydetecting an organophosphatase enzyme in a sample suspected to containan organophosphatase and a phosphatase comprising (a) contacting thesample with a compound of the formula II:

wherein R¹¹-R¹⁴ are selected from the group consisting of H and groupsor atoms other than H, X⁴-X⁹ are independently O or S, n and m are 0 or1 but m and n cannot be 0 simultaneously, and R¹⁵-R²⁴ can be H or anysubstituent so long as the compound of formula II upon hydrolysisprovides a fluorescent product; (b) collecting the fluorescent product;(c) measuring the fluorescence of a fluorescent product formed duringthe contacting; and (d) correlating the measured fluorescence with theactivity of the organophosphatase enzyme.
 69. A method for specificallyand selectively detecting and/or measuring the activity of anorganophosphatase enzyme immobilized on a support comprising: (a)contacting the support with a compound of the formula II:

wherein R¹¹-R¹⁴ are selected from the group consisting of H and groupsor atoms other than H, X⁴-X⁹ are independently O or S, n and m are 0 or1 but m and n cannot be 0 simultaneously, and R¹⁵-R²⁴ can be H or anysubstituent so long as the compound of formula II upon provides afluorescent product; (b) collecting the fluorescent product; (c)measuring the fluorescence of a fluorescent product formed during thecontacting; and (d) correlating the measured fluorescence with theactivity of the organophosphatase enzyme.